Heterologous expression of ACS-CODH variants and characterisation of the cdh operon in Methanococcus maripaludis

ABSTRACT

Methanogenic archaea are anaerobic microorganisms capable of converting CO2 and H2 into methane, making them promising candidates for CO2 valorisation and bio-based fuel production. The ACS/CODH enzyme complex, which catalyses carbon-carbon bond formation, is central to this metabolism and therefore a key target for metabolic engineering. This thesis investigates the physiological role of the cdh operon in Methanococcus maripaludis and explores strategies for heterologous expression of ACS/CODH variants in M. maripaludis and Methanosarcina acetivorans. Optical density assays, gas chromatography and NMR spectroscopy were used to assess the impact of cdh deletion on growth and metabolism. Heterologous expression efforts involved CRISPR/Cas12-based genomic integration in M. maripaludis and plasmid-based expression in M. acetivorans. (Delta)cdh M. maripaludis strains exhibited growth in acetate-deficient medium, suggesting a partial deletion strain due to polyploidy. Heterologous expression efforts produced intermediate CRISPR donor plasmids and successfully amplified ACS/CODH variant fragments from a variety of Methanobacteriota, providing a foundation for future studies. These findings highlight the physiological complexity and biotechnological potential of ACS/CODH in methanogens, informing strategies for the engineering of synthetic pathways for CO2 valorisation and bio-based fuel production.